manufacturing techniques, were a mass production of ADEX foils can be realized. Therefore, these ADEX foils can be integrated into reusable Lab-on-chip devices enabling the recording of multiple bilayers. We assume that this technique can improve electrical recordings in reconstituted lipid bilayer membranes.
Figure 4.1: Capacitance and resistance of Teflon and ADEX foils. Intact Teflon or ADEX foils (A) or foils with approx. 100 μm large aperture (B) were sealed between two chambers of a planar lipid bilayer set-up.
After filling both chambers with 100 mM KCl solution and after generating a stable DPhPC bilayer over the apertures in B, the current responses to a voltage ramp (lower trace) were measured. The initial current jump at the onset of the ramp is a measure for the capacitance. The resistance can be obtained from the slop (red line) of the current during the voltage ramp.
larger pore diameter to the side of the film that was exposed to the UV light in the lithographic preparation.
To examine the application of ADEX septa in planar lipid bilayer (PLB) recordings, we used foils with apertures of approx. 100 μm diameter; for comparison, apertures with similar dimensions were also generated by electric sparks in Teflon (Fig. 4.1). Both septa were positioned in conventional cuvettes for a vertical bilayer set-up (Bartsch et al., 2012). Planar lipid bilayers of DPhPC phospholipid were then generated with the painting or pseudo painting/air bubble technique (Müller et al., 1967; Braun et al., 2014b). After establishing a stable bilayer, the capacitance (Cp) and resistance (Rp) of single pores were measured in both systems from voltage ramps (Fig. 4.1). In case of ADEX septa, the Cp value was 57 pF (mean 57 ± 1 pF, n=4) (Fig. 4.1B) in a pore with a mean diameter of 102 ± 2 μm. To obtain the specific capacitance of the lipid bilayer (Cm) in the pore, the mean Cc value measured in Fig. 4.1A was subtracted from Cp and the remaining value normalized to the mean area of the pore. In this way, we can estimate a mean specific membrane capacitance Cm of 0.3 ± 0.01 μF/cm2 and 0.6 ± 0.02 μF/cm2 for bilayers in pores of approx. 100 μm diameter in ADEX and Teflon foils, respectively. The Cm value in Teflon is compatible with data reported for other bilayer systems including Teflon as septum (Winterstein et al., 2018; Sugawara & Hirano, 2005). The specific capacitance of the membrane in the ADEX foil is lower. One possible explanation for the lower capacitance could be due to the different geometry of the bilayer
Figure 4.2: Imaging of apertures in Teflon and ADEX foils. The upper row shows a top view on single apertures in Teflon (A) and ADEX foils (B&C). Illustrated are overlays of bright field images (grey) with fluorescent images (green) from FITC in the pore. The central rows show the corresponding side view of the pores reconstructed from confocal scans of fluorescein fluorescence; the borders in the Teflon foil are indicated by dotted lines. The edges of the ADEX foil are visible from the self-reflection of the 633 nm laser, which does not occur in Teflon. The lower row reports data from individual intensity scans (grey points) and their mean values (red lines; n ≥ 4) for the respective pores. The data were obtained by measuring the grey value along a line at the equator of single pores in a top view perspective. Grey values from different pores were normalized to the same ordinate. Scale bar is valid for all panels.
in the apertures in the ADEX films with a larger rim and a smaller effective bilayer. The results of these experiments indicate that the apertures in ADEX films carry both disadvantages (e.g.
lower resistance) and advantages (e.g. lower specific capacitance) compared to similarly sized apertures in conventional Teflon films. ADEX septa are thus generally suited to host stable, free-standing planar lipid bilayers. Yet, because of inferior electrical properties, ADEX films will not necessarily improve the signal-to-noise ratio in ion channel recordings compared to Teflon foils.
Figure 4.3: Comparative recordings of channel activity in septa from Teflon and ADEX films. (A) Typical channel fluctuations of KcvNTS channel at ±120 mV in symmetrical solution with 100 mM KCl. Data were measured in DPhPC bilayer painted over an aperture in Teflon (open circles) or ADEX (closed circles) foils with apertures of 100 μm and 50 μm respectively. (B) Mean I/V relation (± s.d.) of unitary KcvNTS currents from recordings as in (A) in Teflon (black circles; n=4) or in ADEX foil (open circles; n=3). (C) Mean open probability/voltage (Po/V) relation (mean± s.d.) of KcvNTS channel from recordings as in (B). (D) I/V relations of KcvNTS channel reconstituted in bilayers in ADEX foils as in (A) with apertures of 100 ( ) 50 (△) or 30 (▲) μm diameter; inset: representative current traces at -120 mV for three different apertures in ADEX foil. (E), I/V relations of KcvNTS channel in ADEX foil with 100 μm diameter as in A before (◆) and after (◇) cleaning septum in Acetone.
In subsequent experiments, we compared the functional properties of a reference K+ channel, the small viral protein KcvNTS (Rauh et al., 2018), that has been reconstituted in lipid bilayers formed over ADEX- and Teflon-based septa. Representative recordings of single channel fluctuations in a DPhPC bilayer generated over an approx. 100 μm large hole in Teflon
foil or over approx. 50 μm large hole in ADEX foil are shown in Fig. 4.3 In both cases, we measured the same type of channel activity. At positive voltages, the channel exhibits well-resolved channel openings and closings. At negative voltages of approx. -100 mV, the
unitary openings become increasingly noisy; the latter is caused by a typical fast gating at negative voltages, which cannot be resolved in conventional recording set-up (Rauh et al., 2018). From the unitary channel fluctuations, we constructed the current/voltage relation as well as the open probability/voltage in both recording conditions (Fig. 4.3B&C). A comparison of the data shows that the basic functional features of the K+ channel can be measured in both recording set-ups and independently of the size of the aperture in the ADEX septum (Fig. 4.3B&D). It is also worth mentioning that the insertion of a channel into a bilayer formed over the ADEX septum occurs with the same bias as for a Teflon septum (Winterstein et al., 2018).
To test the stability of recordings in ADEX-based septa, measurements as in Fig. 4.4 were kept for as long as 48 h before they were actively terminated. In all cases, the recordings proceeded without experiencing any instability of the bilayer. This suggests that bilayers formed over ADEX septa are very stable. In a next set of experiments, we further examined the stability of channel recordings in ADEX- and Teflon septa. To this end, we periodically removed the medium in the trans chamber of the bilayer set-up. This operation generally destroys the bilayer over a Teflon septum (not shown). In other cases, the bilayer is first destroyed and then spontaneously reforming during the refilling of the measuring chamber. Since this newly formed bilayer does no longer contain the channel of interest (Fig. 4.4A) the procedure does not fulfill the purpose of a solution exchange.
The situation is very different for lipid bilayers formed over a septum in ADEX foil; the representative example in Fig. 4.4B shows that the chamber could be frequently emptied and refilled without compromising the quality of the bilayer. The results of these experiments demonstrate that lipid bilayer membranes formed over ADEX septa are mechanically much more stable compared to those in Teflon.
ADEX films are also resistant to acetone, which provides the possibility of cleaning
them from lipids and proteins. To test the possibility of reusing ADEX septa for bilayer recordings, we measured channel activity as in Fig. 4.3 in an ADEX film with a 30 μm pore. The septum was then washed for 1 min with acetone, for 5 min with isopropanol and subsequently rinsed twice in distilled water. After painting a new bilayer over the aperture, the same type of K+ channel was reconstituted and measured. The functional data, which are here represented by the unitary I/V relation in Fig. 4.3E, show no difference between the channel performance in fresh or recycled septum. The results of these experiments show that ADEX foils can be easily reused after cleaning with acetone for channel recordings, and provides a critical property for the design and handling of sensor devices based on ADEX foils.
Figure 4.4: Sensitivity of bilayers in Teflon and ADEX septa to solution exchange. Continuous recordings of KcvNTS channel activity at +120 mV in symmetrical solution with 100 mM KCl in Teflon (A) and ADEX (B) foil.
During the recording the solution of the trans chamber was removed and resupplied from a 5000 μl pipette. This procedure resulted in a destruction and reformation of the bilayer. While the original bilayer (left) exhibited an active channel the reformed bilayer (right) did not. The same procedure could be frequently repeated with the ADEX septum (B) without losing the bilayer with the active channel. One cycle of removing (blue bar) and resupplying of the solution (red bar) in B is magnified.